Biomass-based bioenergy is crucial to meet the goal of making cellulosic biofuels cost-competitive with gasoline. Lignocellulosic materials represent an abundant feedstock for cellulosic-biofuel production. A core challenge in converting cellulosic material to biofuels such as ethanol and butanol is the recalcitrance of biomass to breakdown. Because of the complex structure of lignocellulosic biomass, pretreatment is necessary to make it accessible for enzymatic attack. Severe biomass pretreatments are required to release the sugars, which along with by-products of fermentation can create inhibitors in the production of ethanol or butanol, for example. During the pretreatment processes, a range of inhibitory chemicals are formed that include sugar degradation products such as furfural and hydroxymethyl furfural (HMF); weak acids such as acetic, formic, and levulinic acids; lignin degradation products such as the substituted phenolics vanillin and lignin monomers. In addition, the metabolic byproducts such as ethanol, lactate, and acetate also impact the fermentation by slowing and potentially stopping the fermentation prematurely. The increased lag phase and slower growth increases the ethanol cost due to both ethanol production rate and total ethanol yield decreases.
Efficient conversion of lignocellulosic hydrolysates to biofuel requires high-yield production and resistance to industrially relevant stresses and inhibitors. To overcome the issue of inhibition caused by pretreatment processes, there are two approaches, one is to remove the inhibitor after pretreatment from the biomass physically or chemically, which requires extra equipment and time leading to increased costs. A second approach utilizes inhibitor tolerant microorganisms for efficient fermentation of lignocellulosic material to ethanol (Almeida et al., Journal of Chemical Technology and Biotechnology 82, 340-349, 2007).
Two different genes have been identified recently that confer enhanced tolerance to pretreatment inhibitors (Yang et al., Proc. Natl. Acad. Sci. USA 107:10395-400, 2010; Yang et al., BMC Microbiology 10:135, 2010). Microbial ethanol tolerance has been thought to be a complex and likely a multigenic trait (Williams et al., Appl. Microbiol. Biotechnol. 74: 422-432, 2007; Timmons et al., Appl. Microbiol. Biotechnol. 82: 929-939, 2009). As reviewed by Stephanopoulos (Science 315: 801-804, 2007), there has been accumulating evidence that no single gene can endow microbes with tolerance to ethanol and other toxic compounds. To date, little progress has been made in identification of key genetic changes that confer enhanced ethanol tolerance. Global transcription machinery engineering (gTME) is an approach that has improved glucose/ethanol tolerance in Saccharomyces cerevisiae and led to increased productivity (Alper et al., Science 314, 1565-1568, 2006). See also U.S. Published Application 2007/0072194 A1.
In prokaryotic systems, there is increasing evidence for the link between alcohol dehydrogenases and maintenance of cellular redox-balance under ethanol stress conditions. For example, an ethanol adapted strain 39EA of Thermoanaerobacter ethanolicus (formerly Clostridium thermohydrosulfuricum) was found to lack detectable levels of NAD-linked ADH activity as compared to the wild-type strain (Lovitt, R. W. et al., 1988. Ethanol-production by thermophilic bacteria-biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum. J. Bacteriol. 170:2809-2815). Similarly, T. ethanolicus strain 39E H8 adapted to high ethanol levels also lacked activity for the primary alcohol dehydrogenase that is involved in nicotinamide co-factor recycling while increasing the percentage of transmembrane fatty acids (Burdette, D. S. et al., 2002. Physiological function of alcohol dehydrogenases and long-chain (C30) fatty acids in alcohol tolerance of Thermoanaerobacter ethanolicus. Appl. Environ. Microbiol. 68:1914-1918). Thus, mutations in alcohol dehydrogenase genes and redox balance may be beneficial for adaptation to elevated ethanol levels in bacterial strains.
Bacterial systems such as Thermoanaerobacter ethanolicus contain primary and secondary alcohol dehydrogenases with differing co-factor specificities. In C. thermocellum, conflicting biochemical studies suggest that the alcohol dehydroganses are either NADH-specific (15) or capable of utilizing NADH or NADPH (24). Among four Fe-containing alcohol dehydrogenases in C. thermocellum, Cthe0423, a bi-functional aldehyde/alcohol dehydrogenase, is the third most abundant transcript in the cell, while the other alcohol dehydrogenases are transcribed in much lower abundance (6), suggesting that Cthe0423 is the main ethanol dehydrogenase in C. thermocellum. 